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        Agar Plates for Selection of Clones in Bacteria

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        Agar Plates for Selection of Clones in Bacteria 

        Cloning of PCR products

        Stocks:

        LB Agar: Luria Broth after Lennox:

        per Liter

        Tryptone 10 g
        Yeast Extract

        5 g

        Sodium Chloride 5 g
        Bact.  Agar 15 g

         pH 7.0

        Autoclave and store sterile at room temperature 

        Stock solutions:
        Store all frozen in aliquots at -20C (can be stored in dark at 4C but -20 is usually as simple!)

        X-Gal: 20 mg/ml in Dimethylformamide (DMF)
        IPTG: 100 mM
        Ampicillin: 100 ug/ml (micrograms)

        Filter sterilize

        X-Gal is 5-bromo-4-chloro-3-indolyl-beta-D-galactoside. It is hydolysed by. beta galactosidase to form an indigo-type blue-green precipitate. Bacteria with the B-galactosidase gene give blue colonies.
        IPTC is isopropylthio-beta-D-galactoside. It is a false substrate (not cleaved) and inducer of  beta-galactosidase
        Ampicillin is an antibiotic which requires the resistance plasmid in the bacteria

        Final concentrations on plates

        X-gal of 80ug/ml (1:250 dilution of the stock solution, can use half this)
        IPTG 0.05 mM /L (1:200 dilution of stock, can use twice this)

        so to 100 ml LB agar (molten but < 50C, ie you can hold the bottle in your hand without burning), add 100 ul Ampicillin stock, 400 ul X-Gal stock and 500 ul IPTG stock

        Use 20 ml per Petri dish (cover bottom then move once side-to-side to distribute).

         

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